CN114982958A - Targeted modified astaxanthin-loaded cow milk exosome nano preparation and preparation method thereof - Google Patents
Targeted modified astaxanthin-loaded cow milk exosome nano preparation and preparation method thereof Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C7/00—Other dairy technology
- A23C7/04—Removing unwanted substances other than lactose or milk proteins from milk
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C7/00—Other dairy technology
- A23C7/04—Removing unwanted substances other than lactose or milk proteins from milk
- A23C7/046—Removing unwanted substances other than lactose or milk proteins from milk by centrifugation without using chemicals, e.g. bactofugation; re-use of bactofugate
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
- A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
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Abstract
The invention relates to a target-modified astaxanthin-loaded cow milk exosome nano preparation and a preparation method thereof, belonging to the fields of food industry and nano preparations. According to the invention, cow milk exosomes are used as a carrier, amphiphilic distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is used as a connecting structure, hyaluronic acid with a targeting function is modified on the surface of a cow milk exosome phospholipid bilayer membrane, and astaxanthin with biological activity is loaded in an ultrasonic mode to form a stable astaxanthin targeting carrier system. The prepared target modified astaxanthin-loaded cow milk exosome nano preparation has good comprehensive performance: compared with free astaxanthin, the water solubility, the thermal stability, the oxidation resistance and the inhibition effect on cell oxidative damage caused by lipopolysaccharide induction are all obviously improved, and the biocompatibility is good.
Description
Technical Field
The invention relates to a target-modified astaxanthin-loaded cow milk exosome nano preparation and a preparation method thereof, belonging to the fields of food industry and nano preparations.
Background
Astaxanthin is a biologically active substance with xanthophyll, carotenoid, and deep red color, and is mainly present in algae, yeast, and shrimps. The astaxanthin has various health care functions of resisting diabetes, resisting inflammation, regulating immunity and the like, and has wide application prospect in functional foods. In addition, astaxanthin has a very strong antioxidant activity and a very strong singlet oxygen quenching capacity, and is an incredible active oxygen scavenger. Due to the existence of a large number of unsaturated bonds in the structure, the material is unstable in ultraviolet rays and hot acidic and alkaline solutions and is easy to degrade. In addition, astaxanthin exists mainly in the form of esters, and is poor in water solubility, so that the bioavailability of the astaxanthin is limited.
Disclosure of Invention
[ problem ] to
The preparation method of the astaxanthin-loaded targeted modified cow milk exosome nano preparation based on the exosomes from natural cow milk is provided, and the prepared targeted modified astaxanthin-loaded cow milk exosome nano preparation has good comprehensive performance: compared with free astaxanthin, the water solubility, the thermal stability, the oxidation resistance and the inhibition effect on cell oxidative damage caused by lipopolysaccharide induction are all obviously improved, and the biocompatibility is good.
[ solution ]
The invention aims to provide a preparation method of a targeted modified astaxanthin-loaded cow milk exosome nano preparation, which comprises the following steps:
(1) centrifuging milk for the first time to remove fat impurities with larger particles in the milk;
(2) taking the supernatant obtained in the step (1), adjusting the pH value to 4.6, and precipitating protein impurities;
(3) centrifuging the system obtained in the step (2) for the second time, taking supernatant and filtering to remove small-particle precipitated impurities;
(4) centrifuging the filtrate obtained in the step (3) for the third time, and taking the precipitate, namely the cow milk exosome;
(5) fully dissolving and uniformly mixing the cow milk exosomes and a neutral phosphate buffer solution to obtain cow milk exosome dispersion liquid;
(6) preparation of hyaluronic acid-distearoylphosphatidylethanolamine-polyethylene glycol 2000 Wherein the hyaluronic acid is in combination with distearoylphosphatidylethanolamine-polyethylene glycol 2000 The mass ratio of (A) to (B) is 10: 1;
(7) mixing the cow milk exosome dispersion liquid obtained in the step (5) with the hyaluronic acid-distearoylphosphatidylethanolamine-polyethylene glycol obtained in the step (6) 2000 Mixing according to the mass ratio of 20:1, stirring and reacting to obtain the milk exosome dispersion liquid modified by hyaluronic acid;
(8) dissolving astaxanthin in alcohol to obtain an astaxanthin alcohol solution; mixing the hyaluronic acid modified cow milk exosome dispersion liquid obtained in the step (7) with an astaxanthin alcohol solution according to the mass ratio of cow milk exosomes to astaxanthin being (8-50) to 1; performing intermittent ultrasound and repeated incubation on ice under an ice bath condition, and performing incubation to obtain hyaluronic acid modified milk exosome dispersion liquid loaded with astaxanthin;
(9) and (5) centrifuging the hyaluronic acid modified astaxanthin-loaded cow milk exosome dispersion liquid obtained in the step (8) to remove non-embedded astaxanthin, and removing the solvent by a rotary evaporation method to obtain the hyaluronic acid modified astaxanthin-loaded cow milk exosome nano preparation.
In some embodiments, the first centrifugation in step (1) is performed at 10000-13000 g at 10 ℃ for 30 min.
In some embodiments, the second centrifugation in step (3) is performed at 10000-13000 g for 30min at 10 ℃.
In some embodiments, the third centrifugation in step (4) is ultracentrifugation at 135000-150000 g at 10 ℃ for 90 min.
In a preferred embodiment of the present invention, in step (8), the hyaluronic acid-modified milk exosome dispersion obtained in step (7) is mixed with an astaxanthin alcohol solution in a mass ratio of milk exosomes to astaxanthin of 10: 1.
As a preferred embodiment of the present invention, the step (6) specifically includes: activating carboxyl in hyaluronic acid; then, distearoylphosphatidylethanolamine-polyethylene glycol 2000 Dissolving in dimethylformamide to obtain distearoylphosphatidylethanolamine-polyethylene glycol 2000 And activityThe hyaluronic acid solution after being dissolved is prepared from hyaluronic acid and distearoylphosphatidylethanolamine-polyethylene glycol 2000 Is mixed according to the mass ratio of 10:1, stirred for 12 hours at room temperature, dialyzed and purified to obtain hyaluronic acid-distearoylphosphatidylethanolamine-polyethylene glycol 2000 。
As a preferred embodiment of the present invention, a method of activating carboxyl groups in hyaluronic acid: hyaluronic acid is dissolved in water, and N-hydroxy thiosuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide are added to activate carboxyl groups in hyaluronic acid.
In a preferred embodiment of the invention, in the step (7), the reaction is carried out for 12-24 hours under stirring at 4 ℃.
As a preferred embodiment of the present invention, in step (8):
intermittent ultrasonic: the intermittent ultrasound and ice incubation circulation under the ice bath condition is specifically as follows: carrying out intermittent ultrasonic treatment for 2 minutes under the ice bath condition with the ultrasonic power of 200-250W, wherein the intermittent ultrasonic treatment program is carried out according to the opening time of 15 s/the closing time of 15 s;
incubation on ice: after intermittent sonication, incubation was continued on ice for 2 minutes;
the above batch sonication, ice incubation steps were cycled 3 times.
As a preferred embodiment of the present invention, the ultrasonic power in step (8) is 200W.
As a preferred embodiment of the present invention, in step (8), the incubation is carried out at 37 ℃ for 1 hour.
The second purpose of the invention is to provide the target modified astaxanthin-loaded cow milk exosome nano-preparation prepared by the method.
The third purpose of the invention is to provide the application of the target modified astaxanthin-loaded cow milk exosome nano preparation in preparing food and health care products.
[ advantageous effects ]
(1) The invention uses cow milk exosome which has a phospholipid bilayer structure similar to a cell membrane and is secreted by cells as a carrier, and uses amphiphilic distearoylphosphatidylethanolamine-polyethylene glycol 2000 As a connecting structure, hyaluronic acid with a targeting function is modified on the surface of a cow milk exosome phospholipid bilayer membrane, and astaxanthin with biological activity is loaded in an ultrasonic mode to form a stable astaxanthin targeting carrier system.
(2) According to the invention, hyaluronic acid is modified on the surface of the cow milk exosome, so that the targeting ability of the cow milk exosome as a nano-carrier can be improved; by distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 Realizes the modification of hyaluronic acid on cow milk exosomes and overcomes the technical problems of poor compatibility and difficult compounding of hyaluronic acid and cow milk exosomes.
(3) The preparation method of the exosome targeted astaxanthin-delivery nano preparation based on the natural cow milk source provided by the invention has the advantages that the prepared targeted modified astaxanthin-loaded cow milk exosome nano preparation has good comprehensive performance: compared with free astaxanthin, the water solubility, the thermal stability, the oxidation resistance and the inhibition effect on cell oxidative damage caused by lipopolysaccharide induction are obviously improved, and the biological compatibility is good.
(4) Compared with free astaxanthin, the astaxanthin coated by the cow milk exosomes has improved effects of protecting RAW264.7 cells from antioxidant damage caused by LPS induction and reducing the production of proinflammatory factors in the cells, and a targeting carrier system formed after further modification by hyaluronic acid has better antioxidant and anti-inflammatory activities compared with the loading of single cow milk exosomes.
(5) The research of the invention finds that the cow milk exosome modified by hyaluronic acid can be used as an ideal delivery system of astaxanthin, can effectively improve the bioactivity of astaxanthin and increase the stress resistance of astaxanthin.
Drawings
FIG. 1 is a transmission electron micrograph of bovine milk exosomes obtained in step S4 of example 1 of the present invention.
FIG. 2 is a transmission electron micrograph of astaxanthin-loaded bovine milk exosomes obtained in step S5 of example 1 of the present invention.
FIG. 3 is a transmission electron micrograph of the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes obtained in example 2 of the present invention.
FIG. 4 is a transmission electron micrograph of cow's milk exosomes isolated by differential centrifugation alone according to comparative example 1 of the present invention.
FIG. 5 is a graph showing the comparison of the embedding ratios of astaxanthin measured in example 3 of the present invention.
Fig. 6 is a uv spectrum of astaxanthin obtained after treatment of free astaxanthin and hyaluronic acid-modified bovine milk exosomes obtained in step S6 of example 2 of the present invention.
Fig. 7 is a thermogravimetric analysis of free astaxanthin, astaxanthin-loaded bovine milk exosomes of example 1 of the present invention, hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes of example 2.
FIG. 8 is a graph showing the effect of different concentrations of the bovine-derived extracellular vesicles (bovine milk exosomes) of example 1 on cell viability according to the present invention.
FIG. 9 is a graph showing the effect of different concentrations of astaxanthin-loaded bovine milk exosomes of example 1 of the present invention on cell viability.
Fig. 10 is a graph of the effect of different concentrations of hyaluronic acid modified astaxanthin-loaded bovine milk exosomes of example 2 of the present invention on cell viability.
FIG. 11 is a graph showing a comparison of the protective fluorescence intensity of free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) against Lipopolysaccharide (LPS) -induced inflammatory cell models in accordance with the present invention under the same astaxanthin concentration conditions.
FIG. 12 is a graph comparing the inhibition of tumor necrosis factor in Lipopolysaccharide (LPS) -induced inflammatory cell models by free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) in the present invention under the same astaxanthin concentration conditions.
Detailed Description
The present application is not limited to the specific conditions and details in the following embodiments. The various specific features may be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, all possible combinations are not further described herein. Any person skilled in the art can make simple modifications and substitutions within the technical scope of the present invention described in the present invention according to his own circumstances, and the simple modifications are within the protective scope of the present invention. In practicing the present invention, various alternatives to the embodiments of the invention described herein may be employed. The following detailed description of the embodiments of the present invention is provided for the purpose of illustrating the present invention and is not intended to limit the reagent or apparatus used in the present invention.
The invention is further illustrated by the following specific examples.
Purchase channel and product information for distearoylphosphatidylethanolamine-polyethylene glycol 2000 in example 2: is purchased from Shanghai Michelin Biochemical technology Co., Ltd, and the purity is more than or equal to 98 percent.
Example 1: preparation method of astaxanthin-loaded cow milk exosome
S1, placing the cow milk in a high-speed centrifuge bottle, and centrifuging for 30min at the rotation speed of 13000g and the temperature of 10 ℃ to remove fat impurities with larger particles in the cow milk;
s2, taking the supernatant centrifuged in the step S1, and adjusting the pH value of the supernatant to 4.6 by using 2mol/L hydrochloric acid solution so as to precipitate protein impurities in the supernatant;
s3, centrifuging the system obtained in the step S2 at 10000g and 10 ℃ for 30min, taking supernatant, and filtering the supernatant by a filter membrane with the pore size of 0.22 mu m to remove small-particle precipitates such as cell debris and the like;
s4, ultracentrifuging the filtered clear liquid obtained in the step S3 at 135000g and 10 ℃ for 90min, taking the precipitate, namely the exosome (cow milk exosome) from cow milk, and completely dissolving the precipitate in a neutral phosphate buffer solution to obtain the dispersion liquid of the cow milk exosome.
S5, mixing the dispersion liquid of the cow milk exosomes obtained in the step S4 with an ethanol solution of astaxanthin according to the mass ratio of the cow milk exosomes to the astaxanthin of 10:1, carrying out intermittent ultrasonic treatment for 2 minutes by adopting an ultrasonic load method under the conditions of ultrasonic power of 200-250W and ice bath, carrying out the intermittent ultrasonic treatment program according to the opening time of 15S/closing time of 15S, and then continuously incubating for 2 minutes on ice; and (3) circulating the intermittent ultrasonic incubation step for 3 times to obtain the milk exosome dispersion liquid loaded with the astaxanthin. The astaxanthin-loaded bovine milk exosome dispersion was incubated at 37 ℃ for 1 hour to restore the stability of bovine milk exosome membranes.
Centrifuging (3000 Xg, 5min, 25 ℃) the cow milk exosome dispersion liquid loaded with astaxanthin to remove non-embedded astaxanthin, and removing ethanol by a rotary evaporation method at 37 ℃ to obtain the cow milk exosome nano preparation loaded with astaxanthin.
Example 2: preparation method of hyaluronic acid modified astaxanthin-loaded cow milk exosomes
Steps S1-S4 of example 2 are the same as steps S1-S4 of example 1;
s5 preparation of hyaluronic acid-distearoylphosphatidylethanolamine-polyethylene glycol 2000 : first, hyaluronic acid (900mg, 30. mu. mol) was completely dissolved in distilled water, then N-hydroxysuccinimide (40.0. mu. mol) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (50.0. mu. mol) were added to give a mixed solution, and stirring was continued at low temperature for 4 hours to activate carboxyl groups in hyaluronic acid, and then distearylphosphatidylethanolamine-polyethylene glycol was added 2000 Dissolving distearoylphosphatidylethanolamine-polyethylene glycol in Dimethylformamide (DMF) 2000 Mixing with activated hyaluronic acid solution according to hyaluronic acid and distearoylphosphatidylethanolamine-polyethylene glycol 2000 Mixing at a mass ratio of 10:1, stirring at room temperature for 12 hours, transferring the obtained viscous solution into a 3500 Dalton dialysis bag, dialyzing for 48 hours, and freeze-drying to obtain hyaluronic acid-distearoylphosphatidylethanolamine-polyethylene glycol 2000 ；
S6, mixing the dispersion of cow milk exosome obtained in the step S4 with the hyaluronic acid-distearoylphosphatidylethanolamine-polyethylene glycol obtained in the step S5 2000 Mixing according to the mass ratio of 20:1, stirring for 12-24 h at 4 ℃, and obtaining the milk exosome dispersion liquid modified by hyaluronic acid through self-assembly reaction;
s7, mixing the hyaluronic acid modified cow milk exosome dispersion liquid obtained in the step S6 with an astaxanthin ethanol solution according to the mass ratio of cow milk exosomes to astaxanthin being 10: 1; carrying out intermittent ultrasonic treatment for 2 minutes by adopting an ultrasonic load method under the conditions of ultrasonic power of 200-250W and ice bath, wherein the intermittent ultrasonic treatment procedure is carried out according to the condition of opening for 15 s/closing for 15s, and then continuously incubating for 2 minutes on ice; and (3) circulating the intermittent ultrasonic incubation step for 3 times to obtain the hyaluronic acid modified astaxanthin-loaded cow milk exosome dispersion liquid. The hyaluronic acid modified astaxanthin-loaded bovine milk exosome dispersion was incubated at 37 ℃ for 1 hour to restore the stability of bovine milk exosome membranes.
Centrifuging (3000 Xg, 5min, 25 ℃) the hyaluronic acid modified milk exosome dispersion liquid loaded with astaxanthin to remove non-embedded astaxanthin, and removing ethanol by a rotary evaporation method at 37 ℃ to obtain the hyaluronic acid modified milk exosome nano preparation loaded with astaxanthin.
Example 3: optimization of hyaluronic acid modified astaxanthin-loaded cow's milk exosomes-influence of cow's milk exosomes to astaxanthin mass ratio on astaxanthin embedding rate
On the basis of example 2, the mass ratio of the cow milk exosomes to the astaxanthin in step S7 is adjusted, i.e. the cow milk exosomes modified by hyaluronic acid and the astaxanthin are mixed according to the mass ratio of 10:1 and 50:1 respectively, so as to obtain two hyaluronic acid modified astaxanthin-loaded cow milk exosome nano-preparations respectively.
As shown in fig. 5, the astaxanthin encapsulation efficiency (74%) was higher in the condition of the cow milk exosome to astaxanthin mass ratio of 10:1 than in the condition of the astaxanthin mass ratio of 50:1 (54%).
Comparative example 1: cow's milk exosomes separated only by differential centrifugation
A method for extracting cow 'S milk exosomes, referring to example 1, except that step S2 is omitted, i.e. cow' S milk exosomes obtained by differential centrifugation alone are separated.
The test method comprises the following steps:
transmission electron microscopy characterization: 20. mu.L of the bovine milk exosomes obtained in step S4 of example 1, the astaxanthin-loaded bovine milk exosomes obtained in step S5 of example 1, and the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes obtained in example 2 were mixed togetherThe cow milk exosomes obtained by the differential centrifugation method and the cow milk exosomes obtained by the differential centrifugation method in the comparative example 1 are respectively dripped into a copper mesh, and the cow milk exosomes are subjected to negative staining by using 2% uranyl acetate and observed under a transmission electron microscope.
Solubility test：
Mixed solution 1: mixing 1mg astaxanthin with 10. mu.L of hyaluronic acid-modified bovine milk exosomes obtained in step S6 of example 2, vortexing thoroughly for 10 minutes and incubating for 30min, after which 1 ml ddH was added to the above solution 2 And O. Mixed solution 2: 1mg of astaxanthin was dissolved in 1 ml of ddH 2 And (4) in O. Standing the two mixed solutions on ice for 30min, and centrifuging at 5000rpm for 5min to remove undissolved astaxanthin. The astaxanthin concentration was calculated by measuring the UV-visible absorption spectrum in the range of 250-700nm using a UV-visible spectrometer to evaluate the solubility of astaxanthin in water.
The embedding rate of astaxanthin is as follows:
continuously carrying out ultrasonic treatment on the hyaluronic acid modified astaxanthin-loaded cow milk exosome nano preparation to be detected for 30min to release the astaxanthin loaded in cow milk exosomes, then extracting by using dichloromethane/methanol (2:1, v/v), and measuring an ultraviolet-visible absorption spectrum in the range of 250-700nm by using an ultraviolet visible spectrometer to obtain the astaxanthin concentration, thereby determining the astaxanthin embedding rate.
And (3) testing thermal stability:
a Discovery TGA 550 instrument (TA, New Castle, USA) was used to perform thermogravimetric analysis (TGA) to characterize the thermal stability of free astaxanthin, astaxanthin-loaded bovine milk exosomes, hyaluronic acid modified astaxanthin-loaded bovine milk exosomes under the test conditions: the temperature range is 30 to 700 ℃ in nitrogen atmosphere, and the heating rate is 30 ℃ per minute.
Cell viability characterisation: RAW264.7 cells were plated at 1X 10 per well 5 The cells were inoculated in a 96-well plate at a density, incubated for 24 hours to allow the cells to adhere to the wall, the medium was removed, and cow's milk exosomes (example 1) and astaxanthin-loaded cows were added at concentrations of (0, 1, 2, 2.5, 5, 7.5, 10. mu.g/mL) as quantified by cow's milk exosome concentrationMilk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) were incubated for 24 hours, 20 μ L of thiazole blue at a concentration of 5mg/mL was added after the incubation was ended, and the absorbance value was measured at 570nm after shaking for 10min, and the absorbance value at a concentration of 0mg/mL of bovine milk exosomes was set as the cell viability value of 100%.
Intracellular reactive oxygen species level assessment: RAW264.7 cells were plated at 1X 10 per well 5 Individual cell densities were seeded in twelve-well plates. According to the astaxanthin concentration quantification, free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) were dissolved in the medium, and after 24 hours of incubation, the medium was removed and replaced with a medium containing 1 μ g/mL of Lipopolysaccharide (LPS), and the medium alone served as a control group. After 24 hours of treatment, cells were stained with 0.5mL of dichlorodihydrofluorescein diacetate (DCFH-DA, EX ═ 500nm, EM ═ 525nm) for 30 minutes. Immediately after washing three times with PBS, the cells were observed under a fluorescence microscope, and the results of fluorescence intensity exhibited by the cells were analyzed by imagine J software.
Evaluation of proinflammatory factor secretion level in cell: RAW264.7 macrophages were dosed at 1X 10 per well 5 The density of individual cells was inoculated in a twelve-well plate and incubated for 24 hours to allow the cells to adhere to the cell plate, the amount of astaxanthin was quantified in accordance with the same astaxanthin concentration, fresh medium containing astaxanthin alone, astaxanthin-loaded bovine milk exosomes (example 1), hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) was replaced and incubated for 24 hours, after the medium was removed, the medium was replaced with a medium containing lipopolysaccharide 1. mu.g/mL and then incubated for 24 hours, and the intracellular expression level of intracellular tumor necrosis factor (TNF-. alpha.) was measured using a biochemical kit.
And (3) performance comparison:
microscopic morphology: as shown in fig. 1, the bovine milk exosomes obtained in step S4 of example 1 exhibited a cup or sphere shape, with a size of about 100 nm; as shown in fig. 2, the astaxanthin-loaded bovine milk exosomes obtained in step S5 of example 1 exhibited spherical shapes, increasing in size to about 145 nm; as shown in fig. 3, the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes obtained in example 2 still exhibited spherical shapes with a size of about 165 nm; as shown in fig. 4, the cow's milk exosomes of comparative example 1 isolated by differential centrifugation alone were reduced in purity and reduced in density under electron microscopy.
Solubility: as shown in FIG. 6, the ultraviolet spectrum showed a characteristic absorption peak of astaxanthin at about 480nm in the wavelength range of 250-700 nm. The hyaluronic acid-modified bovine milk exosomes obtained in example 2 showed a higher absorption peak after acting on astaxanthin than free astaxanthin under the same astaxanthin content, demonstrating that the hyaluronic acid-modified bovine milk exosomes can greatly improve the solubility of astaxanthin in water.
Thermal stability: as shown in the thermogravimetric analysis curve of fig. 7, both the astaxanthin-loaded bovine milk exosomes (example 1) and the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) exhibited superior thermal stability compared to free astaxanthin: the free astaxanthin has higher weight loss at about 300 ℃, which shows that the thermal stability of the free astaxanthin is poor above 300 ℃; the astaxanthin-loaded cow milk exosome (example 1) and the hyaluronic acid modified astaxanthin-loaded cow milk exosome (example 2) have small weight loss at 20-700 ℃, and the heat stability at 20-700 ℃ is excellent. Based on the above, the astaxanthin-loaded cow milk exosome (example 1) and the hyaluronic acid modified astaxanthin-loaded cow milk exosome (example 2) can be applied to a thermal processing process below 700 ℃, and the application scene is wider than that of free astaxanthin.
Cell viability assay showed: the cow milk exosome (example 1) with the concentration of 1-10 mug/mL has no inhibition effect on cell viability, so that the cow milk exosome has good biocompatibility and can be used as a safe delivery carrier. The astaxanthin-loaded bovine milk exosomes (example 1) and the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) showed a tendency to increase and then decrease in cell viability with increasing bovine milk exosome concentration, and the analysis revealed that both the astaxanthin-loaded bovine milk exosomes (example 1) and the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) were effective in promoting cell proliferation at bovine milk exosome concentrations ranging from 1 μ g/mL to 5 μ g/mL. Preferably, for the cow milk exosomes loaded with astaxanthin (example 1), when the cow milk exosome concentration is 1-2.5 μ g/mL, the cell viability is significantly improved compared with the blank group without the cow milk exosomes, and especially when the cow milk exosome concentration is 2.5 μ g/mL, the cell viability is improved to the highest extent compared with the blank group without the cow milk exosomes, and the improvement ratio reaches 134%. Preferably, for the hyaluronic acid modified astaxanthin-loaded cow milk exosome (example 2), when the cow milk exosome concentration is 2-5 μ g/mL, the cell viability is improved by more than 10% compared with a blank group without adding cow milk exosomes, and especially when the cow milk exosome concentration is 5 μ g/mL, the cell viability is improved to the highest extent and the improvement ratio reaches 111% compared with the blank group without adding cow milk exosomes.
Intracellular reactive oxygen species level: comparison of the protective fluorescence intensity of free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) against Lipopolysaccharide (LPS) -induced inflammatory cell models at the same astaxanthin concentration is shown in fig. 11. Compared with the blank group, the group induced only by lipopolysaccharide shows the highest fluorescence intensity, which indicates that the intracellular oxidative damage is increased and the active oxygen is increased; the fluorescence intensity after treatment with free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) was reduced compared to the lipopolysaccharide-induced group, indicating a reduction in active oxygen content. Of these, example 2, which has a fluorescence intensity of as low as 42% as possible, most closely approached the blank (30%), showed the strongest antioxidant properties, indicating that the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes have a more effective protection against lipopolysaccharide-induced cell damage.
Intracellular proinflammatory factor secretion levels: the inhibition of tumor necrosis factor in Lipopolysaccharide (LPS) -induced inflammatory cell models by free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) at the same astaxanthin concentration is shown in fig. 12. The lipopolysaccharide-only-induced group showed the highest intracellular expression level of intracellular tumor necrosis factor (TNF-. alpha.) compared to the blank group. After the astaxanthin-loaded cow milk exosome (example 1) and the hyaluronic acid modified astaxanthin-loaded cow milk exosome (example 2) are treated, the intracellular expression level of intracellular tumor necrosis factor (TNF-alpha) is obviously reduced compared with that of a lipopolysaccharide induction group, wherein the intracellular expression level of the intracellular tumor necrosis factor (TNF-alpha) of the hyaluronic acid modified astaxanthin-loaded cow milk exosome group of the example 2 is as low as 104pg/mL and is close to that of a blank group (90pg/mL), which shows that the inhibition effect on the intracellular proinflammatory factor is more remarkable, and the hyaluronic acid targeting effect is more prominent.
Claims (10)
1. A preparation method of a target-modified astaxanthin-loaded cow milk exosome nano preparation is characterized by comprising the following steps:
(1) centrifuging milk for the first time to remove fat impurities with larger particles in the milk;
(2) taking the supernatant obtained in the step (1), adjusting the pH value to 4.6, and precipitating protein impurities;
(3) centrifuging the system obtained in the step (2) for the second time, taking supernate and filtering to remove small-particle precipitate impurities;
(4) centrifuging the filtrate obtained in the step (3) for the third time, and taking the precipitate, namely the cow milk exosome;
(5) fully dissolving and uniformly mixing the cow milk exosomes and a neutral phosphate buffer solution to obtain cow milk exosome dispersion liquid;
(6) preparation of hyaluronic acid-distearoylphosphatidylethanolamine-polyethylene glycol 2000 Wherein the hyaluronic acid is in combination with distearoylphosphatidylethanolamine-polyethylene glycol 2000 The mass ratio of (A) to (B) is 10: 1;
(7) mixing the cow milk exosome dispersion liquid obtained in the step (5) with the hyaluronic acid-distearoylphosphatidylethanolamine-polyethylene glycol obtained in the step (6) 2000 Mixing according to the mass ratio of 20:1, stirring and reacting to obtain the milk exosome dispersion liquid modified by hyaluronic acid;
(8) dissolving astaxanthin in alcohol to obtain an astaxanthin alcohol solution; mixing the hyaluronic acid modified cow milk exosome dispersion liquid obtained in the step (7) with an astaxanthin alcohol solution according to the mass ratio of cow milk exosomes to astaxanthin being (8-50) to 1; performing intermittent ultrasound and repeated incubation on ice under an ice bath condition, and performing incubation to obtain hyaluronic acid modified milk exosome dispersion liquid loaded with astaxanthin;
(9) and (4) centrifuging the hyaluronic acid modified astaxanthin-loaded cow milk exosome dispersion liquid obtained in the step (8) to remove non-embedded astaxanthin, and removing the solvent by a rotary evaporation method to obtain the hyaluronic acid modified astaxanthin-loaded cow milk exosome nano preparation.
2. The method according to claim 1, wherein in step (8), the hyaluronic acid-modified bovine milk exosome dispersion obtained in step (7) is mixed with an astaxanthin alcohol solution in a mass ratio of bovine milk exosomes to astaxanthin of 10: 1.
3. The method according to claim 1, characterized in that step (6) comprises in particular: activating carboxyl in hyaluronic acid; then, distearoylphosphatidylethanolamine-polyethylene glycol 2000 Distearoylphosphatidylethanolamine-polyethylene glycol dissolved in dimethylformamide 2000 Mixing with activated hyaluronic acid solution according to hyaluronic acid and distearoylphosphatidylethanolamine-polyethylene glycol 2000 Is mixed according to the mass ratio of 10:1, stirred for 12 hours at room temperature, dialyzed and purified to obtain hyaluronic acid-distearoylphosphatidylethanolamine-polyethylene glycol 2000 。
4. The method according to claim 3, wherein the method for activating carboxyl groups in hyaluronic acid comprises: hyaluronic acid is dissolved in water, and N-hydroxy thiosuccinimide and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide are added to activate carboxyl groups in the hyaluronic acid.
5. The method according to claim 1, wherein the stirring reaction in step (7) is carried out at 4 ℃ for 12-24 h.
6. The method according to claim 1, wherein in step (8):
intermittent ultrasonic: the intermittent ultrasound and ice incubation circulation for multiple times under the ice bath condition specifically comprises the following steps: carrying out intermittent ultrasonic treatment for 2 minutes under the ice bath condition with the ultrasonic power of 200-250W, wherein the intermittent ultrasonic treatment program is carried out according to the opening time of 15 s/the closing time of 15 s;
incubation on ice: after intermittent sonication, incubation was continued on ice for 2 minutes;
the above batch sonication, ice incubation steps were cycled 3 times.
7. The method of claim 6, wherein the ultrasonic power in step (8) is 200W.
8. The method according to claim 1, wherein in step (8), the incubation is performed at 37 ℃ for 1 hour.
9. The targeted modified astaxanthin-loaded bovine milk exosome nano-formulation prepared by the method of any one of claims 1-8.
10. The application of the targeted modified astaxanthin-loaded cow milk exosome nano preparation in preparation of food and health care products according to claim 9.
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